A cracking buffer is used to crack open the bacterium, releasing its DNA (both genomic and plasmid), which is electrophoresed on an agarose gel. The genomic DNA being Mbs in length migrates very slowly while the plasmid DNA move much faster. Plasmid DNA containing insert move slower than those without any; thus possibly positive clones can be identified.

5x Cracking buffer

Sucrose 25 g

sDDW 40 ml

(Dissolve sucrose at 65 deg C)

5 M NaOH 5 ml

10% SDS 2.5 ml

Add sDDW to 50 ml final volume

Take 1 ml aliquot and add 20-40 ul normal 6X or 10X DNA gel loading buffer (or just bromophenol blue; the cracking buffer is dense enough for DNA to fall down into the well) before use. The buffer, with dye, can be kept for less than month (alkaline condition).

Method

Pick single colony with sterile toothpick (as many colonies as necessary to get desired result).

Bacterial colonies of about 1mm size can directly be cracked, right after picking from the plate (no overnight culture needed). To do so, pick the colony with a tooth-pick, and resuspend it in 20 ul LB medium or just water (by vigorously rotating and scraping the pick against wall of the tube). Add cracking buffer to that 20 ul of bacterial suspension. Put the pick into a plastic tube with LB and antibiotics for overnight culture (or just inoculate an LB/antibiotic agar plate at specific position).

RNAse A may be added to the cracking buffer (this will decrease the background smear over the lane that arises from bacterial RNA). You can simply use the buffer P1 (containing RNAse) of plasmid preparation kits (5ul cracking buffer with the dye, 15ul bacteria, 5ul P1).